Mutations in the ATP-binding cassette transporter, ABCA1, have recently been found to cause Tangier Disease, a condition associated with low HDL and cholesterol accumulation in macrophage foam cells. ABCA1 stimulates phospholipid efflux to apoA-1, while cholesterol efflux may occur as a secondary event. ABCA1 gene expression is increased in response to cellular cholesterol loading, due to increased transcription mediated by LXRs, sterol-activated nuclear receptors. However, the underlying mechanisms of physiological regulation, involving the generation of specific oxysterol metabolites in macrophages, are poorly understood. The goals of the proposal are to investigate the molecular mechanisms mediating and regulating macrophage cholesterol efflux to apoA-I and HDL, and the relationship of cholesterol efflux to foam cell formation and atherogenesis. In Aim 1, we will test the hypothesis that the interaction of apoA-I with ABCA1 leads to formation of phospholipid/apoA-1 complexes that then stimulate cholesterol efflux from a distinct region of plasma membrane. The mode of interaction of apoA-I with ABCA1 will be investigated by mutagenesis of ABCAl, the cholesterol efflux pathway stimulated by ABCA1 activity will be further characterized, and genomic and proteomics methods will be employed to identify novel molecules involved in cholesterol efflux. In Aim 2, we will examine the hypothesis that cholesterol 27-OHase (Cyp27) plays a major role in generating oxysterol activators of ABCA1 in macrophage foam cells; these studies will employ samples from patients with genetic deficiency of Cyp27 who develop xanthomas and premature atherosclerosis. In Aim 3, we will test the hypothesis that ABCA1 has an anti-atherogenic role, both as a result of its ability to stimulate cholesterol efflux to apoA-I (a macrophage function), and also by stimulating HDL formation (a non-macrophage function). Transgenic mice with inducible expression of ABCA1 in macrophages or liver will be prepared in order to evaluate the in vivo role of ABCA1. The project relates to the SCOR theme of conducting parallel investigation of human genetic deficiency states and transgenic mouse models as a means of determining the role of specific molecules in atherogenesis, and will involve collaborations with Project 1 (Tabas) and the Clinical, Gene Expression and Pathology Cores.